Primary Supervisor: Dr Ilse Pienaar, University of Birmingham
Secondary supervisor: TBD
PhD project title: Deciphering the Role of Aberrant Mitochondria-Nuclear Crosstalk in Driving Selective Brainstem Neuronal Vulnerability across the Spectrum of Synucleinopathies
University of Registration: University of Birmingham
Synucleinopathies (also called α-synucleinopathies) are a diverse group of neurodegenerative disorders that share a common pathologic lesion composed of pathologic aggregates of fibrillary aggregates of the α-synuclein protein (so-called ‘Lewy Bodies’) that accumulate in the cytoplasm of selective populations of neurons and glia.1 Such disorders include Parkinson's disease (PD), Lewy Body Dementia (LBD), and multiple system atrophy (MSA). Affected patients are clinically characterized by a chronic and progressive decline in motor, cognitive, behavioural and autonomic functions, depending on the distribution of the lesions. Due to clinical overlap, differential diagnosis can be difficult.
Several reports highlighted vulnerability (degeneration followed by cell death) of brainstem-based neuronalpopulations in the synucleinopathies.2 These brain regions control vital functions e.g. respiratory control, sleep and arousal, with MSA patients manifesting impairments affecting these.3-5 The proposed PhD study will intersectionally compare two brainstem nuclei (the pedunculopontine nucleus (PPN) and locus coeruleus (LC)), with certain neurochemical neuronal types within such structures that are subject to synucleinopathy pathology. The genomic profile of vulnerable PPN and LC neurochemical subtypes will then be compared to a neuronal type that is spared. In particular, in α-synucleinopathy brains, some neuronal subtypes show abundant Lewy pathology while this is absent in other neuronal subtypes. In addition, such vulnerable neurons are prone to undergoing apoptosis, while types that do not harbour Lewy Bodies are spared. In this regard, PPN cholinergic and LC noradrenergic neurons show abundant α-synuclein aggregates and eventual cell loss, while GABAergic neurons within both the PPN and LC are mostly spared.6
The overall aim of this project will hence be to identify factors that confer neuronal susceptibility vs resistance to pathogenic mechanisms in syncleinopathy disorders. The genomic interrogation studies will be conducted at single neuron resolution. The neurons will be collected from clinically well-described post-mortem brains of three types of synucleinopathy patients, those diagnosed with PD vs LBD vs MSA, and compared to neurological control patients, to compile a register as to genomic overlap and differences seen between such different synucleinopathy disease types.
The collected neurons will be neurochemically and anatomically confirmed as being as follows: PPN cholinergic (a neuronal type releasing the excitatory neurotransmitter acetylcholine and which degenerates in all synucleinopathies),7-9LC noradrenergic (releasing the catecholamine neuromodulator, noradrenaline, with this neuronal type that also degenerates in the three synucleinpathies to be studied here)10-11 and PPN vs LC GABAergic (neurons releasing GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in the adult brain). Based on our published findings,4we hypothesise that the basis for this selective brainstem neuronal vulnerability rests on intrinsic differences relating to mitochondrial DNA (mtDNA). Multiple studies further highlight mitochondrial (mt) dysfunction in the various synucleinopathy’s pathogenesis incl. mt respiratory chain dysfunctions.12-14 Mitochondria provide cellular energy as adenosine triphosphate (ATP) and fulfil other important cellular functions e.g. controlling programmed cell death. mtDNA encodes core respiratory chain proteins, but most mt proteins are encoded by nuclear DNA. Mito-nuclear interactions facilitate mitochondria-to-nucleus (retrograde) and nucleus-to-mitochondria (anterograde) signals to maintain mt integrity e.g. by regulating gene expression of transcription factor A of mitochondria (TFAM), which packs and protects mtDNA.15
In this PhD student project we aim to resolve the following questions: (1) What interacting mt-nuclear genetic events might initiate and sustain cholinergic vs noradrenergic neuronal brainstem degeneration in synucleinopathies?; (2) to what extent do such events overlap/diverge between the three prominent synucleinopathies, PD, LBD and MSA?; and (3) how do such mechanisms differ from brainstem GABAergic neurons that resist such neurodegeneration?
These questions will be experimentally addressed as follows: Aim 1: Quantify mtDNA copy number (mtCN) and deleted mtDNA molecules in single neurons classed as one of the three distinct types, taken from PD vs LBD vs MSA brains and compared to control post-mortem brains. Mitochondria exist as multiple copies per cell, the number depending on cell type, where mtDNA Copy Number (mtCN) variation associates with numerous diseases and traits.16Coexistence of deleted and wild-type mtDNA molecules in a cell defines ‘heteroplasmy’ ;17 a small ratio of pathological mutations are pervasive, but biochemical malfunctions manifest when deleted mtDNA molecules, driven by intracellular clonal expansion, exceed a threshold (60%).17 We will obtain non-fixed frozen brain tissue blocks (n=12 brain specimens per diagnosis/control) from our partner Brain Banks. Tissue containing the PPN and LC will be cut into thin serial sectionsand mounted on glass slides. After isolating single cholinergic vs noradrenergic vs GABAergic neurons (identified immunohistochemically) from the brainstem nuclei, we will determine mtCN per neuron and proportion deleted mtDNA molecules in the same neuron, using our published methods.4 Aim 2: Characterize mtDNA damage and the response of nuclear genes to the clonal expansion of mtDNA deletions in the synucleinopathies vs control patients in ‘type’-specific single neurons taken from the defined brainstem nuclei. We will perform parallel sequencing of individual neurons’ mtDNA and nuclear/mt transcriptome using the ‘G&T-seq’ method,18,19 allowing us to link levels and locations of mtDNA damage with transcriptomic (RNA sequencing) data of the same neuron. Transcriptome analysis will focus on genes regulating key mt processes, incl. mtDNA maintenance, -biogenesis and mitophagy. Aim 3: Identify nuclear and mt processes associating with synucleinopathy disease severity. We will statistically correlate nuclear/mt transcriptome and mtDNA changes with patient scores obtained from using a well validated indicator of patients’ cognitive functions, namely the Mini–Mental State Examination (MMSE). Specific neuronal populations perish during synucleinopathies, hence we believe this research will identify cell-type specific mitochondrial factors contributing to this vulnerability, to highlight ways to therapeutically target defective systems in affected brains. The findings will further inform on whether distinct differences exist in how nuclear-encoding genetic elements regulate how mtDNA responds to various synucleinopathies, paving the way for therapeutics targeting such mito-nuclear miscommunications, but in a synucleinopathy subtype specific manner.
 Goedert M, et al. (2017). J Parkinsons Dis 7(s1):S51-69;  Brooks DJ, Tambasco N (2016). Mov Disord 31(6):814-29;  Benarroch EE (2019). Clin Auton Res 29(6):549–51;  Bury AG, et al. (2017). Ann Neurol 82(6):1016–21;  Benarroch EE, Schmeichel AM (2001). Ann Neurol 50:640–5;  Hijaz BA, Volpicelli-Daley LA (2020). Mol Neurodegener 15(1):19;  Benarroch EE, et al. (2002). Neurology 59(6):944-6;  Mazere J, et al. (2013). Neuroimage Clin 3:212–7;  Galazky I, et al. (2019). J Neurol 266(9):2244–51;  Lewis SJ, et al. (2012). Neurobiol Dis 46(1):130–6;  Benarroch EE (2003). Cell Mol Neurobiol 23(4-5):519–26;  Benarroch EE (2007). Mov Disord 22:155–61;  Multiple-System Atrophy Research Collaboration (2013). N Engl J Med 369:233–44;  Monzio Compagnoni G, et al. (2018). Biochim Biophys Acta Mol Basis Dis 1864:3588–97;  Foti SC, et al. (2019). Sci Rep 9(1):6559;  Karakaidos P, Rampias T (2020). Life (Basel) 10(9):173;  Castellani CA, et al. (2020). Genome Med 12(1):84;  Stewart JB, Chinnery PF (2015). Nat Rev Genet 16:530–42;  Macauley IC, et al. (2015). Nat Methods 12(6):519–22;  Macauley IC, et al. (2016). Nat Protoc 11(11):2081–103;
BBSRC Strategic Research Priority: Understanding the Rules of Life: Neuroscience and behaviour
Techniques that will be undertaken during the project:
- Human post-mortem brain tissue analysis, including learning key skills in histological validation of specified brain structures. This will include gaining in-depth human brain anatomy expertise. The student will work within a collaborative partnership consisting of neuropathologists, mitochondrial DNA experts and genomic sequencing experts. There is a chance to spend time at the collaborating institutes, which include Imperial College London, the Earlham Institute and the Welcome Trust Centre for Mitochondrial Research at Newcastle University.
- State-of-the-art single cell genomics techniques will be learned during this project. This includes optimal sample preparation and working closely with sequencing collaborating partners to validate the parallel transcriptomics-mitochondrial DNA sequencing approach to be used here.
- The successful candidate will gain expertise in novel bioinformatics, to analyse relevant mitochondrial modifying pathways in a large transcriptome data set and also to link such changes to information on the same cell’s mitochondrial DNA damage (location and extent).
- The student will gain expertise in statistically modelling the genomic data to reflect possible contributions to the progression of the various synucleinopathies.
Contact: Dr Ilse Pienaar, University of Birmingham